RESUMEN
The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical-basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Células Epiteliales/metabolismo , Animales , Polaridad Celular/fisiología , Epitelio/metabolismo , Uniones Intercelulares/metabolismoRESUMEN
Crumbs proteins are evolutionarily conserved transmembrane proteins with essential roles in promoting the formation of the apical domain in epithelial cells. The short intracellular tail of Crumbs proteins are known to interact with several proteins, including the scaffolding protein PALS1 (protein associated with LIN7, Stardust in Drosophila). PALS1 in turn binds to a second scaffolding protein PATJ (PALS1-associated tight junction protein) to form the core Crumbs/PALS1/PATJ complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and development. Here, we investigated the presence and function of PALS1 and PATJ orthologs in C. elegans. We identified MAGU-2 as the C. elegans ortholog of PALS1 and show that MAGU-2 interacts with all three Crumbs proteins and localizes to the apical membrane domain of intestinal epithelial cells in a Crumbs-dependent fashion. Similar to crumbs mutants, magu-2 deletion showed no epithelial polarity defects. We also identified MPZ-1 as a candidate ortholog of PATJ based on the physical interaction with MAGU-2 and sequence similarity with PATJ proteins. However, MPZ-1 is not broadly expressed in epithelial tissues and, therefore, not likely a core component of the C. elegans Crumbs complex. Finally, we show overexpression of the Crumbs proteins EAT-20 or CRB-3 can lead to apical membrane expansion in the intestine. Our results shed light on the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Polaridad Celular/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/metabolismoRESUMEN
ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2 binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single Caenorhabditiselegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo Using CRISPR/Cas9-generated erm-1 mutant alleles, we demonstrate that a PIP2-binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.
Asunto(s)
Actinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto de Actina , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Humanos , Mucosa Intestinal/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Dominios Proteicos , Alineación de SecuenciaRESUMEN
Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or "interactome" networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early-embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.
Asunto(s)
Caenorhabditis elegans/embriología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Mapeo de Interacción de Proteínas , Animales , División Celular , Dominios y Motivos de Interacción de Proteínas , Proteoma , Técnicas del Sistema de Dos HíbridosRESUMEN
The Piwi-piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ARN Interferente Pequeño/biosíntesis , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Metilación , Mutación , Precursores de Proteínas/metabolismo , Interferencia de ARN/fisiología , Transgenes/genéticaRESUMEN
The eukaryotic genome is organized in a three-dimensional structure called chromatin, constituted by DNA and associated proteins, the majority of which are histones. Post-translational modifications of histone proteins greatly influence chromatin structure and regulate many DNA-based biological processes. Methylation of lysine 36 of histone 3 (H3K36) is a post-translational modification functionally relevant during early steps of DNA damage repair. Here, we show that the JMJD-5 regulates H3K36 di-methylation and it is required at late stages of double strand break repair mediated by homologous recombination. Loss of jmjd-5 results in hypersensitivity to ionizing radiation and in meiotic defects, and it is associated with aberrant retention of RAD-51 at sites of double strand breaks. Analyses of jmjd-5 genetic interactions with genes required for resolving recombination intermediates (rtel-1) or promoting the resolution of RAD-51 double stranded DNA filaments (rfs-1 and helq-1) suggest that jmjd-5 prevents the formation of stalled postsynaptic recombination intermediates and favors RAD-51 removal. As these phenotypes are all recapitulated by a catalytically inactive jmjd-5 mutant, we propose a novel role for H3K36me2 regulation during late steps of homologous recombination critical to preserve genome integrity.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Recombinación Homóloga/genética , Histona Demetilasas con Dominio de Jumonji/genética , Recombinasa Rad51/genética , Animales , Apoptosis/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/genética , Daño del ADN/genética , ADN Helicasas/metabolismo , Metilación de ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Células Germinativas , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Procesamiento Proteico-Postraduccional/genética , Recombinasa Rad51/metabolismoRESUMEN
During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα-LGN-NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5-GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Dineínas Citoplasmáticas/genética , Animales , Sistemas CRISPR-Cas , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/metabolismo , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular/genética , Dineínas Citoplasmáticas/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Huso Acromático/genéticaRESUMEN
Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin-proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/fisiología , Hidroximetilglutaril-CoA Sintasa/fisiología , Redes y Vías Metabólicas , Ácido Mevalónico/metabolismo , Sumoilación , Animales , Citosol/metabolismo , Humanos , Lisina/metabolismo , Mitocondrias/metabolismo , Modelos Biológicos , Mutación/genética , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas , Transporte de Proteínas , Ubiquitina/metabolismoRESUMEN
BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide. RESULTS: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules. CONCLUSIONS: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.
Asunto(s)
Proteínas de Caenorhabditis elegans/aislamiento & purificación , Caenorhabditis elegans/metabolismo , Guanilato-Quinasas/metabolismo , Especificidad de Órganos , Mapeo de Interacción de Proteínas/métodos , Secuencia de Aminoácidos , Animales , Biotinilación , Proteínas de Caenorhabditis elegans/metabolismo , Técnica del Anticuerpo Fluorescente , Complejos Multiproteicos/aislamiento & purificación , Neuronas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Reproducibilidad de los ResultadosRESUMEN
The development in early 2013 of CRISPR/Cas9-based genome engineering promises to dramatically advance our ability to alter the genomes of model systems at will. A single, easily produced targeting RNA guides the Cas9 endonuclease to a specific DNA sequence where it creates a double strand break. Imprecise repair of the break can yield mutations, while homologous recombination with a repair template can be used to effect specific changes to the genome. The tremendous potential of this system led several groups to independently adapt it for use in Caenorhabditiselegans, where it was successfully used to generate mutations and to create tailored genome changes through homologous recombination. Here, we review the different approaches taken to adapt CRISPR/Cas9 for C. elegans, and provide practical guidelines for CRISPR/Cas9-based genome engineering.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Roturas del ADN de Doble Cadena , Ingeniería Genética/métodos , Recombinación Homóloga/genética , Animales , Caenorhabditis elegans/genética , Marcación de Gen , Genoma , MutagénesisRESUMEN
Cell proliferation and differentiation are regulated in a highly coordinated and inverse manner during development and tissue homeostasis. Terminal differentiation usually coincides with cell cycle exit and is thought to engage stable transcriptional repression of cell cycle genes. Here, we examine the robustness of the post-mitotic state, using Caenorhabditis elegans muscle cells as a model. We found that expression of a G1 Cyclin and CDK initiates cell cycle re-entry in muscle cells without interfering with the differentiated state. Cyclin D/CDK4 (CYD-1/CDK-4) expression was sufficient to induce DNA synthesis in muscle cells, in contrast to Cyclin E/CDK2 (CYE-1/CDK-2), which triggered mitotic events. Tissue-specific gene-expression profiling and single molecule FISH experiments revealed that Cyclin D and E kinases activate an extensive and overlapping set of cell cycle genes in muscle, yet failed to induce some key activators of G1/S progression. Surprisingly, CYD-1/CDK-4 also induced an additional set of genes primarily associated with growth and metabolism, which were not activated by CYE-1/CDK-2. Moreover, CYD-1/CDK-4 expression also down-regulated a large number of genes enriched for catabolic functions. These results highlight distinct functions for the two G1 Cyclin/CDK complexes and reveal a previously unknown activity of Cyclin D/CDK-4 in regulating metabolic gene expression. Furthermore, our data demonstrate that many cell cycle genes can still be transcriptionally induced in post-mitotic muscle cells, while maintenance of the post-mitotic state might depend on stable repression of a limited number of critical cell cycle regulators.
Asunto(s)
Caenorhabditis elegans/genética , Ciclo Celular/genética , Ciclina D/genética , Ciclina D/metabolismo , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/genética , Células Musculares/citología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/metabolismo , Diferenciación Celular , Proliferación Celular , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Replicación del ADN/genética , Regulación del Desarrollo de la Expresión Génica , Células Musculares/metabolismo , Especificidad de Órganos/genéticaRESUMEN
Physical interactions between proteins are essential for biological processes. Hence, there have been major efforts to elucidate the complete networks of protein-protein interactions, or "interactomes", of various organisms. Detailed descriptions of protein interaction networks should include information on the discrete domains that mediate these interactions, yet most large-scale efforts model interactions between whole proteins only. We previously developed a yeast two-hybrid-based strategy to systematically map interaction domains and generated a domain-based interactome network for 750 proteins involved in C. elegans early embryonic development. Here, we expand the concept of Y2H-based interaction domain mapping to the genome-wide level. We generated a human fragment library by randomly fragmenting the full-length open reading frames (ORFs) present in the human ORFeome collection. Screens using several proteins required for cell division or polarity establishment as baits demonstrate the ability to accurately identify interaction domains for human proteins using this approach, while the experimental quality of the Y2H data was independently verified in coaffinity purification assays. The library generation strategy can easily be adapted to generate libraries from full-length ORF collections of other organisms.
Asunto(s)
Sistemas de Lectura Abierta/genética , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteínas/aislamiento & purificación , Mapeo Cromosómico , Biblioteca de Genes , Genoma Humano , Humanos , Unión Proteica , Proteínas/química , Proteínas/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Intermediate filaments (IFs) are major components of the metazoan cytoskeleton. A long-standing debate concerns the question whether IF network organization only reflects or also determines cell and tissue function. Using Caenorhabditis elegans, we have recently described mutants of the mitogen-activated protein kinase (MAPK) SMA-5 which perturb the organization of the intestinal IF cytoskeleton resulting in luminal widening and cytoplasmic invaginations. Besides these structural phenotypes, systemic dysfunctions were also observed. We now identify the IF polypeptide IFB-2 as a highly efficient suppressor of both the structural and functional deficiencies of mutant sma-5 animals by removing the aberrant IF network. Mechanistically, perturbed IF network morphogenesis is linked to hyperphosphorylation of multiple sites throughout the entire IFB-2 molecule. The rescuing capability is IF isotype-specific and not restricted to sma-5 mutants but extends to mutants that disrupt the function of the cytoskeletal linker IFO-1 and the IF-associated protein BBLN-1. The findings provide strong evidence for adverse consequences of the deranged IF networks with implications for diseases that are characterized by altered IF network organization.
Asunto(s)
Proteínas de Caenorhabditis elegans , Filamentos Intermedios , Animales , Filamentos Intermedios/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Intestinos , Citoesqueleto/metabolismoRESUMEN
Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or 'edgetic', alleles. We established a proof of concept with CED-9, a Caenorhabditis elegans BCL2 ortholog. Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Mapeo de Interacción de Proteínas/métodos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Alelos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Proteínas de Unión al Calcio/genética , Genes de Helminto , Genotipo , Modelos Moleculares , Fenotipo , Proteínas Represoras/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
To provide accurate biological hypotheses and elucidate global properties of cellular networks, systematic identification of protein-protein interactions must meet high quality standards.We present an expanded C. elegans protein-protein interaction network, or 'interactome' map, derived from testing a matrix of approximately 10,000 x approximately 10,000 proteins using a highly specific, high-throughput yeast two-hybrid system. Through a new empirical quality control framework, we show that the resulting data set (Worm Interactome 2007, or WI-2007) was similar in quality to low-throughput data curated from the literature. We filtered previous interaction data sets and integrated them with WI-2007 to generate a high-confidence consolidated map (Worm Interactome version 8, or WI8). This work allowed us to estimate the size of the worm interactome at approximately 116,000 interactions. Comparison with other types of functional genomic data shows the complementarity of distinct experimental approaches in predicting different functional relationships between genes or proteins
Asunto(s)
Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Mapeo de Interacción de Proteínas/métodos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Línea Celular , Humanos , Unión Proteica , Programas InformáticosRESUMEN
Reorganization of the plasma membrane and underlying actin cytoskeleton into specialized domains is essential for the functioning of most polarized cells in animals. Proteins of the ezrin-radixin-moesin (ERM) and Na+/H+ exchanger 3 regulating factor (NHERF) family are conserved regulators of cortical specialization. ERM proteins function as membrane-actin linkers and as molecular scaffolds that organize the distribution of proteins at the membrane. NHERF proteins are PDZ-domain containing adapters that can bind to ERM proteins and extend their scaffolding capability. Here, we investigate how ERM and NHERF proteins function in regulating intestinal lumen formation in the nematode Caenorhabditis elegans. C. elegans has single ERM and NHERF family proteins, termed ERM-1 and NRFL-1, and ERM-1 was previously shown to be critical for intestinal lumen formation. Using CRISPR/Cas9-generated nrfl-1 alleles we demonstrate that NRFL-1 localizes at the intestinal microvilli, and that this localization is depended on an interaction with ERM-1. However, nrfl-1 loss of function mutants are viable and do not show defects in intestinal development. Interestingly, combining nrfl-1 loss with erm-1 mutants that either block or mimic phosphorylation of a regulatory C-terminal threonine causes severe defects in intestinal lumen formation. These defects are not observed in the phosphorylation mutants alone, and resemble the effects of strong erm-1 loss of function. The loss of NRFL-1 did not affect the localization or activity of ERM-1. Together, these data indicate that ERM-1 and NRFL-1 function together in intestinal lumen formation in C. elegans. We postulate that the functioning of ERM-1 in this tissue involves actin-binding activities that are regulated by the C-terminal threonine residue and the organization of apical domain composition through NRFL-1.
RESUMEN
Auxin-inducible degradation is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the auxin-inducible degradation system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in Caenorhabditiselegans, as previously reported in human cells. We tested the depletion of a series of proteins with various subcellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for 5 out of 6 proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for 2 of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system, which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that the degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the auxin-inducible degradation system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.
Asunto(s)
Caenorhabditis elegans , Ácidos Indolacéticos , Animales , Caenorhabditis elegans/metabolismo , Humanos , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ProteolisisRESUMEN
Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Receptores Notch/fisiología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Drosophila , Femenino , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Jagged-1 , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas de la Membrana/genética , Proteínas Serrate-Jagged , Transducción de Señal , Vulva/fisiologíaRESUMEN
The positive selection of V(alpha)14 invariant (i)NKT cells in mice requires CD1d-mediated Ag presentation by CD4(+)CD8(+) thymocytes. Maturation of newly selected iNKT cells continues in the periphery and also involves CD1d expression. CD1d molecules acquire Ags for presentation in endosomal compartments, to which CD1d molecules have access through an intrinsic CD1d-encoded tyrosine motif and by association with the class II MHC chaperone, invariant chain. In this study, we report the generation of mice in which all CD1d is replaced by CD1d-enhanced yellow fluorescent fusion protein (EYFP). CD1d-EYFP molecules are stable, present lipid Ags, and have near normal subcellular distribution. CD1d-EYFP molecules mediated positive selection of V(alpha)14 iNKT cell precursors at decreased efficiency, caused a delay in their terminal maturation, and did not invoke V(alpha)14iNKT cell effector function as wild-type CD1d could. Using these mice, we show that the intrinsic CD1d-encoded sorting motif mediates thymic selection and activation of V(alpha)14 iNKT cells by professional APCs, while for peripheral terminal differentiation the intrinsic CD1d sorting motif is dispensable.
Asunto(s)
Presentación de Antígeno , Antígenos CD1d/metabolismo , Células T Asesinas Naturales/citología , Secuencias de Aminoácidos , Animales , Proteínas Luminiscentes , Ratones , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Timo/citologíaRESUMEN
Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.